Analyte assay using particulate labels

Abstract

Method for specific detection of one or more analytes in a sample. The method includes specifically associating any one or more analytes in the sample with a scattered-light detectable particle, illuminating any particle associated with the analytes with light under conditions which produce scattered light from the particle and in which light scattered from one or more particles can be detected by a human eye with less than 500 times magnification and without electronic amplification. The method also includes detecting the light scattered by any such particles under those conditions as a measure of the presence of the analytes.

What is claimed is:  
     
       1. A method for specific detection of one or more analytes in a sample, comprising the steps of: 
       specifically binding any said one or more analytes in said sample with at least one scattered-light detectable particle of a size between 1 and 500 nm inclusive,  
       illuminating any said particles bound with said analytes with non-evanescent wave light under conditions which produce scattered light from said particle and in which light scattered from one or more said particles can be detected by a human eye with less than 500 times magnification and without electronic amplification, and  
       detecting said light scattered by any said particles under said conditions as a measure of the presence of said one or more analytes,  
       wherein said illuminating and said detecting are on opposite sides of a solid phase surface.  
     
     
       2. The method of claim  1 , wherein said particle has a size which produces a specific colored light when observed by said human eye and illuminated with polychromatic light. 
     
     
       3. The method of claim  2  wherein the color of said specific colored light provides a measure of the presence or amount of said one or more analytes. 
     
     
       4. The method of claim  1 , wherein said detecting comprises measurement of the intensity of scattered light as a measurement of the presence or amount of said one or more analytes. 
     
     
       5. The method of claim  1 , wherein said detecting comprises measurement of the color of scattered light as a measurement of the presence or amount of said one or more analytes. 
     
     
       6. The method of claim  1 , wherein said particle has a composition which produces a specific colored light when observed by said human eye and illuminated with polychromatic light. 
     
     
       7. The method of claim  1 , wherein said particles are bound to a solid phase bound analyte. 
     
     
       8. The method of claim  1 , wherein said particles are suspended in a liquid phase during said detecting step. 
     
     
       9. The method of claim  1 , wherein said analyte is bound to a solid phase. 
     
     
       10. The method of claim  9 , further comprising a wash step to remove unbound analyte. 
     
     
       11. The method of claim  1 , wherein said analyte is free in liquid solution. 
     
     
       12. The method of claim  1 , wherein said one or more analytes are specifically bound onto a microarray or array chip comprising discrete areas each of which may contain said one or more analytes. 
     
     
       13. The method of claim  12 , wherein said illuminating comprises scanning with a light beam. 
     
     
       14. The method of claim  13 , wherein said scanning is performed by moving an illuminating beam. 
     
     
       15. The method of claim  13 , wherein said scanning is performed by moving the sample. 
     
     
       16. The method of claim  12 , wherein separate spatially addressable sites on said array are separately illuminated. 
     
     
       17. The method of claim  12 , wherein the array is illuminated with a broad light beam. 
     
     
       18. The method of claim  1 , wherein said light is polychromatic light. 
     
     
       19. The method of claim  1 , wherein a monochromatic light illumination source is used to provide said light. 
     
     
       20. The method of claim  1 , wherein said method comprises providing a plurality of different particles each having a different visual appearance when observed by said human eye. 
     
     
       21. The method of claim  1 , wherein said particles are used in a homogeneous assay and wherein two or more particles bound to said one or more analytes are brought sufficiently close together so that the light scattering property of at least one particle is altered by particle—particle perturbations, wherein said alteration is a measure of the presence or amount of said one or more analytes. 
     
     
       22. The method of claim  1 , wherein said particles are used in an assay and wherein two or more particles bound to said one or more analytes are brought sufficiently close together so that the light scattering property of the two or more particles can be resolved from single particles and said light scattering is a measure of the presence or amount of said one more analytes. 
     
     
       23. The method of claim  1 , wherein said particles are used in a homogeneous assay and wherein two or more particles bound to said one or more analytes are brought sufficiently close together so that the light scattering property of the two or more particles can be resolved from single particles and said light scattering is a measure of the presence or amount of said one or more analytes. 
     
     
       24. The method of claim  1 , wherein said particles are used in a homogeneous assay and wherein two or more particles that are held in close proximity to one another are caused to be separated so that the light scattering property of any one particle is altered, wherein said alteration is a measure of the presence or amount of said one or more analytes. 
     
     
       25. The method of claim  1 , wherein said particles are used in a homogeneous assay and wherein two or more particles are linked together by one or more molecular interactions, wherein the molecular interaction holding the particles together is disrupted so that one or more particles is released from the molecular interaction, wherein said released particle or particles is a measure of the presence or amount of said one or more analytes. 
     
     
       26. The method of claim  1 , wherein said particle is a gold or silver particle. 
     
     
       27. The method of claim  1 , wherein said light is directed toward said particle by a prism or other light guide system. 
     
     
       28. The method of claim  1 , wherein said particle is spherical and has a size between 10 and 200 nm inclusive. 
     
     
       29. The method of claim  1 , wherein said particle is spherical and has a size between 20 and 200 nm inclusive. 
     
     
       30. The method of claim  1 , wherein said particle is spherical and has a size between 40 and 120 nm inclusive. 
     
     
       31. The method of claim  1 , wherein said particle is spherical and has a size between 80 and 120 nm inclusive. 
     
     
       32. The method of claim  1 , wherein said particle spherical and has a size between 1 and 10 nm inclusive. 
     
     
       33. The method of claim  1 , wherein said particle spherical and has a size between 11 and 40 nm inclusive. 
     
     
       34. The method of claim  1 , wherein said particle spherical and has a size between 100 and 250 nm inclusive. 
     
     
       35. The method of claim  1 , wherein said particle spherical and has a size greater than 250 nm. 
     
     
       36. The method of claim  1 , wherein said particle is spherical. 
     
     
       37. The method of claim  1 , wherein said particle is ellipsoidal. 
     
     
       38. The method of claim  1 , wherein said particle is asymmetric. 
     
     
       39. The method of claim  1 , wherein said particle comprises a multiple particle aggregate. 
     
     
       40. The method of claim  1 , wherein said illuminating and said detecting are on opposite sides of a solid phase surface. 
     
     
       41. The method of claim  1  or  40 , wherein said illuminating is from below a solid phase surface and said detecting is from above said solid phase surface. 
     
     
       42. The method of claim  1 , wherein said illuminating and said detecting are on the same side of a solid phase surface. 
     
     
       43. The method of claim  42 , wherein said illuminating and said detecting are from above said solid phase surface. 
     
     
       44. The method of claim  42 , wherein said detecting is at an angle to said surface outside the cone of the illuminating light and outside the angle of reflection of the illuminating light. 
     
     
       45. The method of claim  1 , wherein said detecting is at angles outside the angles of incident, transmitted, refracted and reflected light from the illuminating light. 
     
     
       46. The method of claim  1 , wherein said detecting is at right angles to the surface of said sample. 
     
     
       47. The method of claim  1 , wherein said detecting is at angles outside the envelope of forward scattered light. 
     
     
       48. The method of claim  1 , wherein said analyte comprises a protein or peptide. 
     
     
       49. The method of claim  48 , wherein said protein comprises a cell surface constituent. 
     
     
       50. The method of claim  48 , wherein said protein comprises a receptor. 
     
     
       51. The method of claim  48 , wherein said protein comprises an antibody. 
     
     
       52. The method of claim  1 , wherein said one or more analytes comprises one or more analytes on a cell surface, in a cell lysate, or in a chromosome preparation. 
     
     
       53. The method of claim  1 , wherein said analyte comprises an antigen. 
     
     
       54. The method of claim  1 , wherein said analyte comprises a pharmaceutical agent. 
     
     
       55. The method of claim  1 , wherein said one or more analytes comprise combinatorial molecules. 
     
     
       56. The method of claim  1 , wherein said analyte comprises a hormone. 
     
     
       57. The method of claim  1 , wherein said analyte comprises a lipid or carbohydrate. 
     
     
       58. The method of claim  1 , wherein said analyte comprises a nucleic acid molecule. 
     
     
       59. The method of claim  1 , wherein two or more different nucleic acid probe molecules are each bound to said particle, and each said probe will bind to a different target sequence in a nucleic acid analyte. 
     
     
       60. The method of claim  1 , wherein a binding agent is attached to said particles. 
     
     
       61. The method of claim  60 , wherein said binding agent is adsorbed to said particle. 
     
     
       62. The method of claim  60 , wherein said binding agent is covalently attached. 
     
     
       63. The method of claim  60 , wherein said binding agent is attached to a base material. 
     
     
       64. The method of claim  63 , wherein a plurality of different base materials are used. 
     
     
       65. The method of claim  60 , wherein said binding agent is proteinaceous. 
     
     
       66. The method of claim  65 , wherein said binding agent is an antibody. 
     
     
       67. The method of claim  65 , wherein said binding agent is a receptor. 
     
     
       68. The method of claim  60 , wherein said binding agent is a nucleic acid. 
     
     
       69. The method of claim  60 , wherein said binding agent is selected from the group consisting of antigens, lectins, carbohydrates, biotin, avidin, streptavidin, and pharmaceutical agents. 
     
     
       70. The method of claim  60 , wherein said particles are coated with a stabilizing layer. 
     
     
       71. The method of claim  70 , wherein said stabilizing layer is a thin metal coating. 
     
     
       72. The method of claim  1 , wherein said particles comprise coated particles. 
     
     
       73. The method of claim  72 , wherein the coating of said coated particles comprises a stabilizing coating. 
     
     
       74. The method of claim  73 , wherein the coating comprises polymers, proteins, peptides, hormones, antibodies, nucleic acids, or receptors. 
     
     
       75. The method of claim  73 , wherein said stabilizing layer is a thin metal coating. 
     
     
       76. The method of claim  1 , wherein the particles detected are detected following metallographic enlargement. 
     
     
       77. The method of claim  76 , wherein said metallographic enlargement is with gold or silver. 
     
     
       78. The method of claim  1 , further comprising refractive index enhancement of the sensitivity and specificity of detection of said particles. 
     
     
       79. The method of claim  1 , further comprising filtering light through one or more narrow band pass filters. 
     
     
       80. The method of claim  1 , further comprising filtering scattered light through a polarization filter or a bandpass filter or both. 
     
     
       81. The method of claim  1 , wherein said detecting comprises magnification with a microscope 2 to 500 times. 
     
     
       82. The method of claim  81 , wherein said magnification is 10 to 100 times. 
     
     
       83. The method of claim  81 , wherein said detecting comprises use of confocal microscopy. 
     
     
       84. The method of claim  1 , wherein said detecting is by eye. 
     
     
       85. The method of claim  1 , wherein said detecting uses a photodetector. 
     
     
       86. The method of claim  85 , wherein said photodetector comprises a photodiode or photodiode array. 
     
     
       87. The method of claim  85 , wherein said photodetector comprises a photomultiplier tube. 
     
     
       88. The method of claim  85 , wherein said photodetector comprises a camera or video camera. 
     
     
       89. The method of claim  85 , wherein said photodetector comprises a charge-coupled device. 
     
     
       90. The method of claim  1 , wherein said illuminating light is non-polarized. 
     
     
       91. The method of claim  1 , wherein said illuminating light is polarized. 
     
     
       92. The method of claim  1 , wherein said illuminating light is from a filament lamp source. 
     
     
       93. The method of claim  1 , wherein said illuminating light is from a discharge lamp source. 
     
     
       94. The method of claim  1 , wherein said illuminating light is from a laser. 
     
     
       95. The method of claim  1 , wherein said illuminating light is from a light emitting diode. 
     
     
       96. The method of claim  1 , wherein said illuminating light is pulsed. 
     
     
       97. The method of claim  1 , wherein said illuminating light is constant. 
     
     
       98. The method of claim  1 , wherein said illuminating light is coherent. 
     
     
       99. The method of claim  1 , wherein said illuminating light is noncoherent. 
     
     
       100. The method of claim  1 , wherein said analyte is in a sample selected from the group consisting of water, urine, blood, sputum, and tissue. 
     
     
       101. The method of claim  1 , wherein said one or more analytes comprises a plurality of different analytes and said one or more particles comprises a plurality of different particles. 
     
     
       102. The method of claim  1 , wherein said one or more analytes are in a medium comprising particulate matter that scatters light non-specifically to the same or greater intensity as the fluorescence from 10 −9  M fluorescein. 
     
     
       103. The method of claim  102 , wherein said particulate matter in said medium scatters light non-specifically to the same or greater intensity as the fluorescence from 10 −8  M fluorescein. 
     
     
       104. The method of claim  102 , wherein said particulate matter in said medium scatters light non-specifically to the same or greater intensity as the fluorescence from 10 −7  M fluorescein. 
     
     
       105. The method of claim  1 , wherein said particles are mixed composition particles, comprising two or more different materials. 
     
     
       106. The method of claim  1 , further comprising providing spatial filtering to reduce non-specific background light. 
     
     
       107. The method of claim  1 , further comprising providing cutoff filters, narrow bandpass filters, or both to reduce non-specific background light. 
     
     
       108. The method of claim  1 , wherein scattered light from individual particles is detected. 
     
     
       109. The method of claim  1 , wherein scattered light from a plurality of particles is detected. 
     
     
       110. The method of claim  1 , wherein said particle has a size that produces a specific intensity level when illuminated under said conditions. 
     
     
       111. The method of claim  1 , wherein said particle has a size that produces a specific polarization when illuminated under said conditions. 
     
     
       112. The method of claim  1 , wherein said particle has a size that produces a specific angular dependency to said scattered light. 
     
     
       113. The method of claim  1 , wherein said particle has a composition that produces a specific intensity level when illuminated under said conditions. 
     
     
       114. The method of claim  1 , wherein said particle has a composition that produces a specific polarization when illuminated under said conditions. 
     
     
       115. The method of claim  1 , wherein said particle has a composition that produces a specific angular dependency to said scattered light. 
     
     
       116. The method of claim  1 , wherein said one or more analytes is in a microtiter plate. 
     
     
       117. The method of claim  1 , wherein said detecting of said one or more analytes is in a flow-based system. 
     
     
       118. The method of claim  1 , wherein said one or more analytes is in a device selected from the group consisting of a flow cytometry apparatus, a microchannel, and a capillary. 
     
     
       119. The method of claim  1 , wherein said particle is a metal or metal-like particle. 
     
     
       120. The method of claim  1 , wherein the light path of said illuminating or scattered light passes through a refractive index matching material that matches the refractive index of adjacent solid material. 
     
     
       121. The method of claim  1 , wherein said method is performed in a homogeneous format. 
     
     
       122. The method of claim  1 , wherein the light path of said illuminating or scattered light passes through an immersion oil that matches the refractive index of adjacent solid material. 
     
     
       123. A method for specific detection of one or more analytes in a sample, comprising the steps of: 
       specifically binding any said one or more analytes in said sample with at least one scattered light detectable particles of a size between 1 and 500 nm inclusive,  
       illuminating any said particles bound with said analytes with non-evanescent wave light under conditions which produce scattered light from said particle and in which light scattered from one or more said particles can be detected by a human eye with less than 500 times magnification and without electronic amplification, and  
       detecting said light scattered by any said particles under said conditions as a measure of the presence of said one or more analytes,  
       wherein said detecting is at an angle to the perpendicular to a solid phase surface less than the critical angle, and wherein said illuminating and said detecting are arranged to reduce non-specific background light thereby allowing said specific detection of said one or more analytes bound with said particles.